Immobilization Matrix Research Articles

Exploiting Silica-Binding and Silica-Forming Proteins as Versatile Tools for One-Step Enzyme Immobilization on Siliceous Materials

Enzyme immobilization has emerged as an essential technique in industrial applications of enzymes. Silica (SiO2) serves as a prominent support material for enzyme immobilization. Recent advancements have led to the development of various silica-binding proteins (SBPs) and silica-forming proteins (SFPs) that are invaluable tools in immobilizing enzymes on siliceous materials in a fast and simple manner. SBPs facilitate the immobilization of enzymes with controlled orientation on silica surfaces, while SFPs enable the biomimetic synthesis and encapsulation of enzymes within silica particles. In this review, we explore recent advances in the use of SBPs and SFPs in enzyme applications. We provide a comprehensive overview of their mechanisms and sequence characteristics relevant to enzyme immobilization. Additionally, we summarize the recombinant production and immobilization procedures for enzymes with SBPs or SFPs. We then categorize the available SBPs and SFPs into naturally occurring and artificially engineered types, presenting recent examples that demonstrate their utilization in enzyme immobilization. Our review highlights the strengths and limitations of various SBPs and SFPs and sheds light on future directions for the development of tailor-made biocatalytic silica.

Bioinspired Nanochitin-Based Porous Constructs for Light-Driven Whole-Cell Biotransformations.

Solid-state photosynthetic cell factories (SSPCFs) are a new production concept that leverages the innate photosynthetic abilities of microbes to drive the production of valuable chemicals. It addresses practical challenges such as high energy and water demand and improper light distribution associated with suspension-based culturing; however, these systems often face significant challenges related to mass transfer. The approach focuses on overcoming these limitations by carefully engineering the microstructure of the immobilization matrix through freeze-induced assembly of nanochitin building blocks. The use of nanochitins with optimized size distribution enabled the formation of macropores with lamellar spatial organization, which significantly improves light transmittance and distribution, crucial for maximizing the efficiency of photosynthetic reactions. The biomimetic crosslinking strategy, leveraging specific interactions between polyphosphate anions and primary amine groups featured on chitin fibers, produced mechanically robust and wet-resilient cryogels that maintained their functionality under operational conditions. Various model biotransformation reactions leading to value-added chemicals are performed in chitin-based matrix. It demonstrates superior or comparable performance to existing state-of-the-art matrices and suspension-based systems. The findings suggest that chitin-based cryogel approach holds significant promise for advancing the development of solid-state photosynthetic cell factories, offering a scalable solution to improve the efficiency and productivity of light-driven biotransformation.

Alkali-mediated Sr incorporation mechanism and binding capacity of alkali aluminosilicate hydrate in geopolymers.

Geopolymers are promising materials for safe immobilisation and disposal of complex radioactive waste streams. This work investigates the effect of Sr incorporation and alkali-activator chemistry on 1) geopolymer chemistry, phase assemblage and nanostructure, 2) chemical binding mechanism of Sr2 + into the aluminosilicate framework of (N,K)-A-S-H gels in geopolymers, and 3) mass transport of Sr2+ during leaching, using high-field solid-state nuclear magnetic resonance spectroscopy and synchrotron-based X-ray absorption spectroscopy measurements. All geopolymers studied comprise a fully polymerised, X-ray amorphous Al-rich (N,K)-A-S-H type gel. Si exists predominantly in tetrahedral Q4(4Al) and Q4(3Al) sites and Al exists in tetrahedral sites, resulting in a net negative charge that is balanced by Na+ and/or K+ in extra-framework sites. Sr2+ was incorporated into extra-framework sites within (N,K)-A-S-H gels, without altering the local structure of the aluminosilicate framework by directly substituting for both Na+ and K+ in charge-balancing sites to form a (N,K,Sr)-A-S-H gel, at loadings equal to or below Sr/Na = 0.005. Above this limit, SrCO3 is formed, and the geopolymers simultaneously chemically bind Sr within a (N,Sr,K)-A-S-H gel, and physically encapsulate excess Sr as SrCO3. These findings have significant implications for use of geopolymers as materials for encapsulation and/or immobilisation of radioactive waste containing 90Sr.

Construction of enzyme-MOFs composite with carbon dots: A strategy to enhance the activity and increase the growth rate of the enzyme-ZIF-8 composite.

Encapsulating enzymes in metal-organic frameworks (MOFs) enhances enzyme protection and improves the accuracy of inhibitor recognition and screening. Zeolitic imidazolate framework-8 (ZIF-8) has been widely used as a host matrix for enzyme immobilization. However, challenges such as the microporous structure and hydrophobicity of ZIF-8, along with the protonation of 2-methylimidazole, hinder the maintenance of activity and the rapid formation of composite. Herein, a new strategy to synthesize novel enzyme-MOFs composite by encapsulating carbon dots (CDs)-modified enzyme and Fe3O4 nanoparticles within ZIF-8 is presented for the first time. The contribution of CDs in enzyme-MOFs composite was investigated. Characterizations reveal that the CDs-modified enzymes compete with imidazole for Zn ions, inducing mesoporous structures that alleviate diffusion limitations. Modification of enzyme with CDs also modulates enzyme-MOFs interfacial interactions, accelerating the formation of composite. Activity evaluation shows that enzyme-MOFs composite (THR@CDs/Fe3O4@ZIF-8) retains 81.76% enzyme activity under harsh conditions and maintains 66.0% of the initial enzyme activity after 10 reuse cycles. This synthesis strategy for the novel enzyme-MOFs composite was proven to be universal. The Km value of THR@CDs/Fe3O4@ZIF-8 (19.32μM) is lower than that of THR/Fe3O4@ZIF-8, indicating that modification with CDs significantly increases the affinity of enzyme. Furthermore, THR@CDs/Fe3O4@ZIF-8 was effectively utilized for enzyme inhibitor recognition and screening. These results demonstrate that the proposed method is a universal approach for rapidly and controllably fabricating enzyme-ZIF-8 composite with elevated activity and exceptional stability, offering promising potential for advanced drug recognition and screening platforms.

Screening of Aluminum-Based MOFs for Effective In Situ Immobilization of Biomolecules.

An effective approach for the immobilization and protection of biological entities is their encapsulation via the in situ synthesis of metal-organic frameworks (MOFs). To ensure the preservation of the bioentities, mild synthetic conditions, including aqueous media and ambient conditions (temperature and pressure), are preferred. In this study, we investigated the synthesis of various aluminum polycarboxylate-based MOFs, including the fumarate, terephthalate, amino-terephthalate, and muconate forms of MIL-53(Al), as well as the MIL-110 and MIL-160 MOF types. The potential as immobilization matrices was then assessed using bovine serum albumin (BSA). Finally, MIL-53(Al)-fum was selected for the encapsulation of a mixture of polysaccharides and more structurally complex bioentities (viruses).

Enzymatic Enantioselective Transesterification Reactions: A Pathway Through Biobased Alternatives

Abstract: The field of biocatalysis has blossomed exponentially over the past decades and revolutionized chemical synthesis, providing greener and sustainable methods for preparing numerous organic molecules at bench and industrial scales and in high stereoselective mode for the chiral ones. However, despite the tremendous progress, researchers still have room to contribute significantly to the field, especially in the valorization of agro-industrial waste to boost the circular (bio)economy. This review summarizes the use of lipases, the most versatile biocatalyst, in enantioselective transesterification reactions. The emphasis is on biobased materials involved in lipase-catalyzed enantioselective transesterification, such as agro-industrial waste for lipases production (isolation source and growth), the use of biobased solvents, renewable acyl donors and biobased materials for enzyme immobilization. We also discuss the perspectives of how to connect the high demand for more robust enzymes and the development of cost-effectiveness enantioselective methods, as well as the challenges to achieving a circular economy.

Benefits of Immobilized Bacteria in Bioremediation of Sites Contaminated with Toxic Organic Compounds.

Although bioremediation is considered the most environmentally friendly and sustainable technique for remediating contaminated soil and water, it is most effective when combined with physicochemical methods, which allow for the preliminary removal of large quantities of pollutants. This allows microorganisms to efficiently eliminate the remaining contaminants. In addition to requiring the necessary genes and degradation pathways for specific substrates, as well as tolerance to adverse environmental conditions, microorganisms may perform below expectations. One typical reason for this is the high toxicity of xenobiotics present in large concentrations, stemming from the vulnerability of bacteria introduced to a contaminated site. This is especially true for planktonic bacteria, whereas bacteria within biofilms or microcolonies have significant advantages over their planktonic counterparts. A physical matrix is essential for the formation, maintenance, and survival of bacterial biofilms. By providing such a matrix for bacterial immobilization, the formation of biofilms can be facilitated and accelerated. Therefore, bioremediation combined with bacterial immobilization offers a comprehensive solution for environmental cleanup by harnessing the specialized metabolic activities of microorganisms while ensuring their retention and efficacy at target sites. In many cases, such bioremediation can also eliminate the need for physicochemical methods that are otherwise required to initially reduce contaminant concentrations. Then, it will be possible to use microorganisms for the remediation of higher concentrations of xenobiotics, significantly reducing costs while maintaining a rapid rate of remediation processes. This review explores the benefits of bacterial immobilization, highlighting materials and processes for developing an optimal immobilization matrix. It focuses on the following four key areas: (i) the types of organic pollutants impacting environmental and human health, (ii) the bacterial strains used in bioremediation processes, (iii) the types and benefits of immobilization, and (iv) the immobilization of bacterial cells on various carriers for targeted pollutant degradation.

Immobilization of Enzymes in Polymeric Materials Based on Polyamide: A Review

The immobilization of enzymes in polyamide-based polymeric materials through covalent bonding is an established technique to stabilize and reuse biocatalysts in industrial processes. Traditionally, enzymes are immobilized using crosslinking agents that activate functional groups on both the support and the enzyme, creating strong bonds that securely anchor the enzyme to the surface. While effective for maintaining enzyme activity over multiple cycles, this method can reduce catalytic efficiency due to rigid binding and involves complex activation steps. Recently, in situ immobilization approaches have emerged as promising alternatives. In this method, enzymes are directly entrapped within the polymer matrix during the synthesis of the polyamide support, such as nylon, simplifying the process and offering enhanced control over enzyme distribution. For instance, studies have demonstrated that in situ immobilization can improve enzyme stability by protecting it within the polymeric network, while reducing production costs and waste. This review explores the ability of polyamide as a support material for immobilization of enzymes, analyzing key techniques, performance across applications, and future strategies to optimize polymer-enzyme interactions for industrial use.

In Situ Ag-Seeded Lamellar Ti3C2 Nanosheets: An Electroactive Interface for Noninvasive Diagnosis of Oral Carcinoma via Salivary TNF-α Sensing.

In the fast-paced quest for early cancer detection, noninvasive screening techniques have emerged as game-changers, offering simple and accessible avenues for precession diagnostics. In line with this, our study highlights the potential of silver nanoparticle-decorated titanium carbide MXene nanosheets (Ti3C2_AgNPs) as an electroactive interface for the noninvasive diagnosis of oral carcinoma based on the prevalence of the salivary biomarker, tumor necrosis factor-α (TNF-α). An in situ reduction was utilized to synthesize the Ti3C2_AgNPs nanohybrid, wherein Ti3C2 acts as the reducing agent, and the resulting nanohybrid was subjected to various characterization techniques to examine the optical, structural, and morphological attributes. The results revealed that spherical AgNPs formed on the surface of Ti3C2 MXene nanosheets by virtue of the low-valent Ti species present in Ti3C2, which facilitated the reduction of AgNO3 to AgNPs. Furthermore, the electrochemical characterization of the nanohybrid-modified screen-printed electrode (Ti3C2_AgNPs/SPE) indicated enhanced heterogeneous electron transfer kinetics. With these encouraging results, the Ti3C2_AgNPs nanohybrid was employed as an immobilization matrix for TNF-α antibodies and applied for electrochemical sensing. Analytical studies of the fabricated immunosensor, conducted by differential pulse voltammetry (DPV), exhibited a broader linear range (1 to 180 pg mL-1), a low limit of detection (0.97 pg mL-1), and high sensitivity (1.214 μA mL pg-1 cm-2) and specificity, even in artificial saliva, indicating its reliability for oral carcinoma diagnosis. Therefore, the Ti3C2_AgNP nanohybrid seems a promising candidate for the effective sensing of TNF-α and could also be explored for other biomarkers.

Assessing immobilization matrices for nuclear effluent treatment: Cs case study

Assessing immobilization matrices for nuclear effluent treatment: Cs case study

An Approach to Monodisperse Polymeric Particles as Matrices for Immobilization of Biosystems

An Approach to Monodisperse Polymeric Particles as Matrices for Immobilization of Biosystems

A Target Responsive Metal Organic Framework Derived Bimetallic Apta-Switch for Reagentless Molecular Recognition.

By integrating iron-cobalt squarate bimetallic metal-organic framework (Fe-Co-SqBMoF) based smart material (SM) with functional DNA (fDNA), we designed a target responsive fDNA@Fe-Co-SqBMoF bioelectrode that exhibits recognition induced switchable response to serve as a reagentless single step electrochemical apta-switch (REA). The construct takes advantage of fDNA ability to bind and concentrate target on the receptor interface, while Fe-Co-SqBMoF@SM multifeatures to serve as an immobilization matrix and a signal generating electrochemical switch. Fe-Co-SqBMoF was introduced to prepare a redox active pencil graphite electrode (PGE), while fDNA (aptamer) was decorated on the receptor PGE to impart specificity and selectivity. The Fe-Co-SqBMoF synthesis was characterized through X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, micro-Raman spectroscopy, dynamic light scattering (DLS), and UV-visible (UV-vis) analysis, while each step of fDNA decoration and bioelectrode fabrication was characterized via field emission scanning electron microscopy (FE-SEM), static water contact angle measurements, cyclic voltammetry (CV), and electrochemical impedance spectroscopy (EIS). To explore the potential application of the fDNA@Fe-Co-SqBMoF bioelectrode, we designed a REA for detection of aflatoxin B1 (AFB1) which permitted the detection of AFB1 in the linear range 0.7-1000 pg/mL with an LOD of 0.54 pg. The practical applicability of an fDNA@Fe-Co-SqBMoF based REA was demonstrated in milk and water samples.

Decorating channel walls in ordered macroporous ZIF-8 with hydrophilic PEG to immobilize lipase for efficient chiral resolution.

The development of efficient immobilization support for the enhancement of enzyme activity and recyclability is a highly desirable objective. Single-crystalline ordered macro-microporous ZIF-8 (SOM-ZIF-8), has emerged as a highly effective matrix for enzyme immobilization, however, the inherent hydrophobic nature limits its further advancement. Herein, we have customized the immobilization of the Pseudomonas cepacia lipase (LP) in the modification-channels of SOM-ZIF-8 by functionalizing the inner surface-properties with polyethylene glycol (PEG) (LP@SOM-ZIF-8-PEG), and significant enhancement of the activity and (thermal, solvent and cyclic) stability can be realized. The incorporation of PEG into SOM-ZIF-8 regulates its inner surface charge and hydrophobic properties, thereby enhancing enzyme loading, facilitating enzyme conformational adjustments, and achieving a uniform dispersion of LP in SOM-ZIF-8-PEG. LP@SOM-ZIF-8-PEG not only demonstrates a pronounced elevation in enzyme loading and activity over LP@SOM-ZIF-8 but also shows an enzyme activity that is impressively three times greater than LP@ZIF-8. It can completely resolve the 1-phenylethanol racemate in 60 min, with a conversion close to 50 % and an enantioselectivity of 99.8 %. After nine cycles of reuse, the LP@SOM-ZIF-8-PEG still holds onto 95 % of its initial activity. The excellent catalytic performance and stability of LP@SOM-ZIF-8-PEG, along with the universality of the PEG modification strategy for other enzymes, make this work promising in industrial applications.

Adsorption Characteristics of Cadmium onto Calcite and Its Agricultural Environmental Relevance

Calcite (CaCO3), a common component of calcium-based fertilizers, has been recognized for its effectiveness as a cadmium (Cd) immobilization agent in the solidification/stabilization (S/S) method. This strategy is a widely used chemical remediation technique aimed at reducing the bioavailability and toxicity of Cd in contaminated soils. This study comprehensively evaluated the potential of calcite for Cd remediation through geochemical analyses, including adsorption isotherms, saturation index, ion concentration changes, and X-ray diffraction (XRD) analysis. Adsorption isotherm experiments indicated that Cd adsorption onto calcite aligns more closely with the Freundlich isotherm model, suggesting a heterogeneous surface with a maximum adsorption capacity of 1.56mgg-1. Based on chemical states result, optimal conditions for CdCO3 precipitation with low Cd concentrations were identified at pH above 7.9. XRD analysis confirmed the formation of Ca0.67Cd0.33CO3 at higher Cd concentrations, indicating that chemisorption is the dominant immobilization mechanism. Additionally, variations in Ca2+ and CO3 2- concentrations supported the substitution of Cd for Ca on the calcite surface. These findings highlight calcite's potential as an effective material for Cd immobilization, providing valuable insights for the developing more sustainable soil remediation strategies.

Electrochemical immunosensor for Bacillus anthracis PA toxin using a polypyrrole-gold nanoparticle/multiwall carbon nanotube sensing platform and cadmium sulphide nanocrystal signal tag.

A sandwich electrochemical immunosensor was proposed for the sensitive detection of Bacillus anthracis protective antigen (B. anthracis PA) toxin based on cadmium sulphide nanocrystals (CdS NCs) and polypyrrole-gold nanoparticle-modified multiwalled carbon nanotubes (PPy-AuNPs/MWCNTs). Herein, PPy-AuNPs/MWCNTs were used as a biocompatible and conducting matrix for immobilization of rabbit anti-PA antibody [RαPA antibody, i.e. capturing antibody (Ab1)] and to facilitate excellent electrical conductivity. PPy-AuNPs/MWCNTs were synthesized through a one-step chemical reaction of pyrrole and Au3+ on the surface of MWCNTs. CdS NCs were employed as a label for covalent binding of monoclonal mice anti-PA antibody [MαPA antibody, i.e. secondary antibody (Ab2)]. When B. anthracis PA toxin was present in an analytical sample, the sandwich immunoassay was formed, and an electrochemical potentiometric stripping analysis (PSA) signal was generated. The PSA response produced during the detection was caused by Cd2+ ions released from CdS NCs upon acid dissolution. The synergistic action of PPy, AuNPs and MWCNTs was responsible for the electrochemical signal amplification in the immunosensor. Under optimal conditions, the developed immunosensor showed a linear range from 100 pg mL-1 to 10 ng mL-1 (R2 = 0.992) with a detection limit of 10 pg mL-1 (S/N = 3) for B. anthracis PA toxin. The developed electrochemical immunosensor also exhibited excellent stability, reproducibility and good specificity. The immunosensor was applied for the analysis of human serum spiked with B. anthracis PA toxin with satisfactory results.

Fabrication of immobilized lipases for medium and long‐chain triacylglycerols preparation through solvent‐free acidolysis

AbstractThe present study aims to fabricate efficient immobilized lipases for medium and long‐chain triacylglycerols preparation through acidolysis. Eighteen novel materials, including five mesoporous materials, four organic groups modified SBA‐15 samples (R‐SBA‐15) and nine macroporous resins, were employed as matrices for lipases immobilization. Lipases of TLL, AOL, RML and CALB were evaluated. Results indicated that TLL exhibited better acidolysis performance and decanoic acid content from 30% to 45% could be obtained; while other lipases (CALB, RML, and AOL) showed poor performance. In addition, macroporous resins of DA‐201, ADS‐17, D3520, and mesoporous silicas of SBA‐15, Al‐MCM‐41, HMS, were suitable for TLL immobilization. TLL@SBA‐15 was selected for acidolysis and the reaction conditions were optimized through single factors study and orthogonal design. The optimum conditions for the acidolysis were: lipase amount 6 wt%, decanoic acid: camellia oil molar ratio 14:1, reaction time 12 h and temperature 50°C. Under these conditions, decanoic acid content at 59.87%, oleic acid content 33.99% and sn‐2 decanoic acid content 7.41% was obtained (the mentioned fatty acids here were the fatty acids combined to oil molecules, not free fatty acids).

METHODOLOGY OF SELECTING CEMENT MATRIX COMPOSITION FOR IMMOBILIZATION OF IRRADIATED URANIUM-GRAPHITE FUEL

The Impulse Graphite Reactor (IGR) is a unique nuclear facility in the world. The core of the research reactor is a stack of uranium-graphite blocks (fuel elements) enriched to 90 wt. % in 235U isotope. As part of the project on conversion of the IGR reactor to low-enriched uranium fuel, the specialists of the Institute of Atomic Energy (IAE) studied the possibility of immobilizing the first core in a cement matrix. Research into the immobilization process included both the formation of technical requirements for the uranium-graphite fuel matrix, determined by the conversion conditions, international and national standards, and the selection of the composition and ratios of the matrix components.The paper presents the results of an analysis of modern achievements in the field of immobilization of radioactive waste and irradiated graphite, the formation of matrix acceptability criteria for the immobilization of highly enriched IGR fuel, and methods for determining whether the matrix properties correspond to the established criteria. The matrix compositions for immobilization of the irradiated fuel of the IGR reactor were selected experimentally, based on such characteristics of the cement slurry as bleed water, viscosity, setting time, uniformity of volume change, homogeneity and strength of the samples. To determine the above characteristics, the methods used to determine the characteristics of cement slurries were used and improved. They have proven their suitability.The requirements used to select the composition of matrices for immobilization of spent uranium-graphite fuel of the IGR reactor turned out to be applicable and sufficiently constructive, and can also be recommended for solving issues of selecting the consistency of matrices for immobilization of other types of RW.

Effect of Sintering Temperature on Microstructure and Properties of Zirconia Ceramics for the Needs of Nuclear Energy

The paper provides experimental results of obtaining high-density ZrO2+3%Y2O3 ceramics, which is promising for use as a matrix for immobilization of HLW. The effect of sintering temperature in the range of 1100...1650 °C on the microstructure of the sintered tablets was studied. Phase composition and microstructure of experimental samples were characterized by XRD and SEM. Grain size distribution analysis was carried out using the "Thixomet" image analyzer. Microhardness was determined using a metallographic complex LECO (USA), an inverted microscope IM-3MET and a hardness tester UIT HVB-30. It was established that increase in the sintering temperature leads to a significant increase in the average grain size (from 85 nm to 1000 nm) and increase in the density of the sintered tablets. Sintering temperature should be at least 1550...1650 ºС to produce high-dense ceramics (97...98 % of theoretical value). Obtained ceramics is characterized by high values of microhardness HV > 12 GPa and crack resistance of 5.5...6.3 MPa·m1/2.

Advances and Challenges in the Development of Immobilized Enzymes for Batch and Flow Biocatalyzed Processes.

The development of immobilized enzymes both for batch and continuous flow biocatalytic processes has gained significant traction in recent years, driven by the need for cost-effective and sustainable production methods in the fine chemicals and pharmaceutical industries. Enzyme immobilization not only enables the recycling of biocatalysts but also streamlines downstream processing, significantly reducing the cost and environmental impact of biotransformations. This review explores recent advancements in enzyme immobilization techniques, covering both carrier-free methods, entrapment strategies and support-based approaches. At this regard, the selection of suitable materials for enzyme immobilization is examined, highlighting the advantages and challenges associated with inorganic, natural, and synthetic organic carriers. Novel opportunities coming from innovative binding strategies, such as genetic fusion technologies, for the preparation of heterogeneous biocatalysts with enhanced activity and stability will be discussed as well. This review underscores the need for ongoing research to address current limitations and optimize immobilization strategies for industrial applications.

Characterization, immobilization and evaluation of anti-Pseudomonas aeruginosa biofilm activity of alginate lyase from marine bacterium, Enterobacter tabaci RAU2C.

Alginate lyases have the potential to be used as a therapeutic agent for P. aeruginosa infections. The present work was focused on the characterization of free and immobilized alginate lyase produced by marine bacteria, Enterobacter tabaci RAU2C isolated previously in the laboratory for alginate lyase production and exploring the potential of alginate lyase as an anti-biofilm agent against the P. aeruginosa biofilm. RAU2C alginate lyase was immobilized using an epoxy-activated curdlan matrix by three different methods. Further, the free and immobilized were characterized for its optimal pH and temperature. The effect of alginate concentration on alginate lyase activity was assessed and the kinetic parameters were evaluated. The anti-biofilm activity of the crude alginate lyase was studied using biofilm inhibition and disruption assays in microtiter plates with crystal violet. The biofilm disruption by RAU2C alginate lyase was also ascertained by microscopic analysis. The immobilization matrix prepared using method 3 had a better binding capacity compared to other methods. Both soluble and immobilized alginate lyase exhibited optimal activity at 37°C and pH 7.0. Km and Vmax of soluble and immobilized alginate lyase were found to be 3.38mg/mL, 22.98mg/mL min and 3.67mg/mL and 26.59mg/mL min respectively. Both microtiter assay and microscopic analysis confirmed the prevention and dispersal of pre-existing biofilms by crude RAU2C alginate lyase, highlighting its potential as an anti-biofilm agent against P. aeruginosa. The study highlights the efficacy of RAU2C alginate lyase as an anti-biofilm agent in controlling P. aeruginosa biofilms.